Oleaginous yeast are defined as those organisms that are naturally capable of oil synthesis and accumulation, wherein oil accumulation ranges from at least about 25% up to about 80% of the dry cell weight. The technology for growing oleaginous yeast with high oil content is well developed (for example, see EP 0 005 277B1; Ratledge, C., Prog. Ind. Microbiol., 16:119-206 (1982)).
And, these organisms have been commercially used for a variety of purposes in the past.
Recently, the natural abilities of oleaginous yeast have been enhanced by advances in genetic engineering, resulting in organisms capable of producing polyunsaturated fatty acids [“PUFAs”], carotenoids, resveratrol and sterols. For example, significant efforts by Applicants' Assignee have demonstrated that Yarrowia lipolytica can be engineered for production of omega-3 and omega-6 fatty acids, by introducing and expressing genes encoding the omega-3/omega-6 biosynthetic pathway (U.S. Pat. Nos. 7,238,482; 7,465,564; 7,550,286; 7,588,931; and 7,932,077; Appl. Publ. Nos. 2009-0093543-A1 and 2010-0317072-A1).
Recombinant production of any heterologous protein is generally accomplished by constructing an expression cassette in which the DNA coding for the protein of interest is placed under the control of a promoter suitable for the host cell. The expression cassette is then introduced into the host cell (i.e., usually by plasmid-mediated transformation or targeted integration into the host genome) and production of the heterologous protein is achieved by culturing the transformed host cell under conditions necessary for the proper function of the promoter contained within the expression cassette. Thus, the development of new host cells (e.g., transformed yeast) for recombinant production of proteins generally requires the availability of promoters that are suitable for controlling the expression of a protein of interest in the host cell.
A variety of strong promoters have been isolated from Yarrowia lipolytica that are useful for heterologous gene expression in yeast, as shown in the Table below.
TABLE 1Characterized Yarrowia lipolytica PromotersPromoterNameNative GeneReferenceXPR2alkaline extracellular U.S. Pat. No. 4,937,189;proteaseEP220864TEFtranslation elongation factor U.S. Pat. No. 6,265,185EF1-α (tef)GPD, GPMglyceraldehyde-3-phosphate-U.S. Pat. Nos. 7,259,255dehydrogenase (gpd),and 7,459,546; U.S. patentphosphoglycerate mutase application Pub. No.(gpm)2011-0059496-A1GPDINglyceraldehyde-3-phosphate-U.S. Pat. No. 7,459,546dehydrogenase (gpd)GPM/chimeric phosphoglycerate U.S. Pat. No. 7,202,356FBAINmutase (gpm)/fructose-bisphosphate aldolase (fba1)FBA, fructose-bisphosphate U.S. Pat. No. 7,202,356FBAIN,aldolase (fba1)FBAINmglycerol-3-phosphateU.S. Pat. No. 7,264,949GPATO-acyltransferase (gpat)YAT1ammonium transporter U.S. patent applicationenzyme (yat1)Pub. Nos. 2006-0094102-A1and 2010-0068789-A1EXP1export proteinU.S. Pat. No. 7,932,077
Additionally, Juretzek et al. (Biotech. Bioprocess Eng., 5:320-326 (2000)) compares the glycerol-3-phosphate dehydrogenase [“G3P”], isocitrate lyase [“ICL1”], 3-oxo-acyl-CoA thiolase [“POT1”] and acyl-CoA oxidase [“POX1”, “POX2” and “POX5”] promoters with respect to their regulation and activities during growth on different carbon sources.
Despite the utility of these known promoters, however, there is a need for new improved yeast promoters for metabolic engineering of yeast (i.e., oleaginous and non-oleaginous) and for controlling the expression of heterologous genes in yeast. Furthermore, possession of a suite of promoters that can be regulated under a variety of natural growth and induction conditions in yeast will play an important role in industrial settings, wherein economical production of heterologous and/or homologous polypeptides in commercial quantities is desirable.
It is believed that promoter regions derived from the Yarrowia lipolytica gene encoding n-alkane-hydroxylating cytochrome P450 [“ALK2”] will be useful in expressing heterologous and/or homologous genes in transformed yeast, including Yarrowia. 